ITMI940192A1 - PURIFIED FORM OF STREPTOGRAMINE, ITS PREPARATION AND PHARMACEUTICAL COMPOSITIONS THAT CONTAIN IT - Google Patents

Description

DESCRIPTION

The present invention relates to a purified form of streptogramins which comprises at least one component of group B of streptogramins associated with at least one "minority" component of group A defined hereinafter by the general formula (II).

Among the known streptogramins, pristinamycin (RP 7293), an antibacterial of natural origin produced by Streptomyces pristinaes-piralis was isolated for the first time in 1955. The pristinamycin marketed under the name of Pyostacin consists mainly of pristinamycin IA and of pristinamycin HA.

Another antibacterial of the streptogramin class: virginiamycin was prepared from Streptomyces virginiae, ATCC 13161 [Antibiotics and Chemotherapy, 5, 632 (1 & 55) J. Virginiamycin (Staphylomycine C ^ R)) consists mainly of the S factor and the factor.

In US patent 3325 359 pharmaceutical compositions have been described which comprise antlbiotic substances which constitute the antibiotic 899: factor S and factor MI.

In the patent application FR 2619 0 (38 the use of components of group A and group B for the treatment of acne has been described.

Antibacterials of natural origin, of the streptogramin class, consist of the mixture of 2 groups of components: components of group B and components of group A, each group having its own antibacterial activity. It has been shown that the association formed by the two groups of components causes a synergy of action which results in increased bacteriostatic and bactericidal activity and the broadening of the spectrum of activity.

In Streptogramine als Modelsysteme fUr den Kationentransport durch Membranen, Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Facultat der Georg-August Universitàt zu Gdttingen, Gdttingen 1979, dans Antibiotics III, 521 (contain family of virgin Inhibycins 1975, Antibiotics, 1975) synergistic component, C. Cocito, Microbiological Reviews, 145-98 (1979) components of groups A and B of streptogramins have been described. J, Preud'Homme, P. Tarridec, et A. Belloc, Bull. Soc. Chim. Fr., 2, 585 (1968) have also described the natural pristinamycin and also the different components that make it up.

All attempts at purified associations of streptogramins systematically involve the majority component of group A [pristynamycin HA (PIIA) j considered as responsible for the activity and synergy of action. Moreover, studies carried out have led to the conclusion that this component that causes a better synergy is important: EP 506561 (page 2).

However, these attempts have never been crowned with success, on the one hand due to the difficulties of industrial preparation and above all due to the fact that purified pristinamycin IIA is a crystallized product whose bioavailability has proved too poor to be able to consider making it the active ingredient of a drug.

From the point of view of the industrial preparation of these products, until now, the available techniques had not allowed to obtain, on a preparative scale, a sufficiently purified form and the production of batches of sufficiently constant and reproducible quality to meet the requirements of the legislations. of certain countries regarding registration.

By way of example, the industrial batches of natural pristinamycin contain, after purification, a quantity of impurities that can reach 203⁄4. The purification attempts employed up to now have systematically led to failures and very often to the degradation of one of the groups of components due to the fact that they are labile products for which numerous operations lead to the opening of the cyclized structure or to the dehydration of the components. of group A. For this reason, in the course of many years, it was considered that an improvement in the degree of purity could not be achieved. In 1988, purification was still considered a problem: J. of Liq. Chromatography, 11 (11), 2367 (1988). Also in 1988, N.K. SHARMA and M.J.O. ANTEUNIS also stated that the separation and purification of the components of virginigimycin was possible for analytical purposes, but was not to be taken into consideration for the production of products, given the difficulties encountered: Bull. Soc. Chim. Belg., 97 (3) 193 (1988).

As a consequence of this situation, the marketing of pristinamiclna (Pyostacine ®) has been definitively limited to certain countries such as France and Belgium. The same happened in

case of virginiamycin (Staphylomycine ^)) marketed only in a limited number of countries as regards the human medicine sector, and also for micamycin whose marketing (limited to Japan) is currently blocked. This has therefore led, in the case of certain populations, to the deprivation of treatment in the case of serious infections caused by gram positive cocci (in particular infections caused by methicillin-resistant staphylococci) or to the deprivation of treatment in the case of sexually transmitted diseases. .

In the field of antibacterials, it is well known to those skilled in the art that allergies or resistances can develop after administration of certain classes of antibiotics [The New England Journal of Medicine, 324 (9), 60: 1 (1991) ^ many strains resistant to Staphylococcus aureus are known in particular in the hospital sector. For this reason, it is extremely useful for the physician to have a wide range of chemically different classes available in order to adapt

the treatment to the particular case of the patient to be treated. The consequence of the absence of commercialization of a particular class can be

serious, if not dramatic, since it

it can result in deprivation of treatment in patients who cannot tolerate other aces of antibiotics.

Thus, the purification tests carried out had always had the aim of eliminating the minority components of streptogramins, these minority components being considered as not indispensable and rather as impurities.

Among the components of group A of natural streptogramins, pristynamycin IIB (PIIB) is a minority component whose proportion by weight is less than 10% in natural pristynamycin and,

more often, it is of the order of magnitude of 8% or even of the order of magnitude of 6% in virginiamycin.

It has now been found, and this constitutes the object of the present invention, that the association constituted by one or more components of group B of general formula:

in which it is a radical with a general formula:

in which R 'is a hydrogen atom or a hydroxyl radical and Y is a hydrogen atom, a methylamine radical or a dimethylamine radical, R is an ethyl radical or when R' represents a hydrogen atom

R may also represent a methyl radical, and R ^ and R ^ represent a hydrogen atom, or

A is a radical of the formula:

1

R is an isobutyl radical, e

is a hydroxyl radical and R ^ is a methyl radical, and consists of one or more "minority" components of group A, with the general formula:

in which R "is a hydrogen atom or a methyl or ethyl radical, is particularly interesting because of its biological activity in vivo.

In fact, the associations according to the invention show a biological action in vivo which is clearly superior to that of the natural product (for example natural virginiamycin or natural pristinamycin) or to that of the associations in which the majority component of group A is involved. and, above all, they have a completely satisfactory bioavailability. Furthermore, these associations can be prepared on a large scale.

It is also possible to access a purified and bioavailable form of a final product with a good level of activity that contains less than 6% impurities.

The product of general formula (II) for which R "is an ethyl radical hereinafter referred to as pristinamycin IIF (PU F) and the product of general formula (II) for which R" is a hydrogen atom hereinafter referred to pristinamycin IIG (PIIG) are novel products that constitute very minority components of streptogramins whose weight proportion is less than 0.5% in natural product batches.

The associations according to the invention are advantageously prepared in proportions of 10/90 up to 90/10 (by weight) or preferably in proportions of 20/80 up to 80/20. They occur in the physical mixture state of the powders, but furthermore, and this constitutes another aspect of the present invention, they occur in the co-precipitate state; or still according to a third aspect of the invention, they present themselves in the co-crystallized state as defined hereinafter.

The present invention also relates to the purified forms consisting of the co-crystallized association of at least one component of group B of general formula (I) with at least one component of group A defined by general formula (II).

Co-crystallization is carried out in the constant stoichiometric ratio of one mole of component (of components) of general formula (I) with

2 moles of component (components) of group A of general formula (II) [this stoichiometric ratio corresponding to the relative proportion of about 43-44 / 57-56 by weight if component A is a product of general formula (I ) for which A ^^ has the structure (la)].

The co-crystallized association according to the invention can be used, alternatively, as a purified and stable antimicrobial agent, and also has an improved in vivo activity and also a good bioavailability, or as a means of purification of a minority component of the streptogramins corresponding to the formula general (II).

In fact, it has never been possible to purify a component of group A of general formula (II) by crystallization; therefore, until now, only chromatographic methods were known for preparing a purified product of group A of general formula (II) and no other purification method was known which would allow the isolation of these products in large quantities.

It has now been pointed out that the component of group A of general formula (II) can be obtained in the pure state by passing through the intermediate of the co-crystallized association defined above. A crude mixture containing at least 30% minority component of group A corresponding to the general formula (II) in solution in an organic solvent such as a ketone (for example, acetone, methylethyl ketone, methyl isobutyl ketone), an ester (for example ethyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate), a chlorinated solvent (for example, methylene chloride, chloroform, dichloro-1,2 ethane), or a nitrile (for example acetonitrile), and added with a component of the group B defined by the general formula (I), leads to obtain a co-crystallized compound in the proportions previously defined. It is understood that the introduced amount of compound of general formula (I) is suitably chosen so that the residual concentration of this product (after co-crystallization) is lower than its solubility in the medium. E. also understood that variations in the respective concentrations of the initial medium in the product of general formula (II) and in the product of general formula (I) do not involve a modification of the co-crystallized compound obtained. The co-crystallized association thus obtained, dissolved in a solvent such as for example methylisobutylketone or dichloroethane, and treated in an acid medium (for example sulfuric acid, hydrochloric acid) allows to obtain, after treatment of the organic phase with a solvent for example hexane, the component minority of group A, purified and exempt from the component of group B.

This association co-crystallizes: a, however, has the advantage of a considerably increased stability, a high degree of purity and above all of allowing an easy industrial realization.

It is clear that this method can also be adapted to the preparation of co-crystallized products with modified derivatives of the natural components of group B of streptogramins and that these co-crystallized substances also fall within the scope of the present invention; likewise, the preparation of the purified forms of the minority components of general formula (II) starting from such co-crystallized products also falls within the scope of the present invention.

A preferred embodiment of the invention relates to an association of pristinamycin IB [as defined above in the case in which it represents a radical of general formula (la) in which Y is a methylamine radical and R 'is a hydrogen atom, and R is an ethyl radical] or virginiamycin SI [as defined above when it represents a radical of general formula (la) for which Y and R 'are hydrogen atoms, and R is an ethyl radical] or a mixture of virginiamycin SI and virginiamycin S4 [as defined above when defined as for virginiamycin SI and R is a methyl radical] with pristinamycin IIB [as defined above with the general formula (II) in which R "is methyl] and containing less 2% of impurities and preferably less than 1% of impurities.

A preferred embodiment of the invention also relates to a purified streptogramin constituted by the association of a component of group B of the streptograms defined by the general formula (I) and by a component of group A of general formula (II) containing a relative proportion of the components of groups B and A in a constant molar ratio of the order of magnitude of 1/2.

According to the invention, the new associations of at least one component of group B of streptogramins of general formula (I) and of at least one component of group A of general formula (II) can be obtained, for example, by preparing the co-crystallized compound such as is defined above. When it is desired to obtain an association in different proportions, the co-crystallized compound thus prepared can be associated with at least one of the components of general formula (I) or with at least one component of group A of general formula (II) previously purified and in a suitable quantity. to obtain the desired proportions, or the component of group A of general formula (II) (or a mixture thereof) can be purified starting from the co-crystallized, therefore it can be mixed in suitable proportions with one or more components of group B of general formula (THE). Alternatively, the combinations according to the invention can be prepared after isolation of the component (s) of group B and one component of group A of general formula (II) starting from the corresponding natural streptogramin, by purification of each of these components, then mixing of the purified components, in the desired proportions as previously defined.

The associations according to the invention can also be made to cover in the desired proportions, starting from a solution of the components of general formula (I) and (II) [or alternatively from a solution of the co-crystallized product and from one of the components of general formula (I ) or (11) 3 i-n methyl isobutyl ketone, in acetone or in methylene chloride, poured into hexane, cyclohexane or water.

The preparation and separation of the components of groups A and B is carried out by fermentation and isolation of the constituents starting from the fermentation must according to or in analogy with the method described by J. Preud'homme et: coll., Bull. Soc. Chim. Fr., vol. 2, 585 (1968), in Antibiot. & Chemother., 5, 632 (1955) or 7, 606 (1957), in Chromatog. Sym., 2nd Brussels, 181 (1962), in Antibiot.

Ann., 728784 (1954-55), in US patent 3299 047

or In Streptogramine als Modelsysterne: fUr den kationentransport durch Membranen, Dlssertatlon zur Erlangung des Ooktorgrades der mathematisch-Naturwissenschaft lichen Facultdt der Georg-August Universitàt zu

Gdttingen, Gdttingen 1979 or as described here

in the examples below. In particular, in the case of pristynamycins, the separation of the components of

Groups A and B is carried out by placing in suspension

crude streptogramin in an organic solvent,

as an acetate (e.g. ethyl acetate) e

then carrying out the filtration or centrifugation of the raw component of group A and the extraction of the component of group B in the middle

aqueous acid and then carrying out a new extraction in clcruTethylmic medium The separation

of the components of groups A and B can also be carried out by acid extraction of a solution of crude streptogramin in methylisobutylketone, then isolation by extraction of the component of group B starting from the aqueous phase

and isolation of the component of group A by precipitation starting from the organic phase.

After separation, purification of the B group components of the streptogramins can be carried out by crystallization in an alcohol

as ethanol, methanol or isopropanol, in an acetate (for example, isopropyl acetate or butyl acetate), in a ketone (for example methylethyl ketone) or in acetonitrile or by chromatography. The purification of the components of group A of general formula (II) can be carried out by chromatography eluting with an acetonitrile-water mixture.

As an alternative, the preparation of the components of groups A and B respectively of general formula (II) and (I) is carried out as described in the French patent application 2689 518, by separate fermentation according to the following stages:

- first stage (optional), mutagenesis on a non-selective streptogramin producing microorganism, and - second stage, selection of the selective microorganisms.

Non-selective microorganisms, in general,

are Actinomycetes and Fungi. The starting microorganisms that can be used in the process are, in particular, non-selective producing microorganisms of

a streptogramin selected from the group including pristinamycin, virginiamycin, micamycin, ostreogricin.

viridogrisein, vernamycin and etamycin. By way

exemplary, non-selective microorganisms which

can be used are mentioned here below

following the table.

ANTIBIOTIC MICROORGANISMS

MUSHROOMS

Micromonospora sp. vernamycin

STREPTOMYCES

S. alborectus virginiamycin S. griseus (NRRL2426) viridogriseina S. lavendulae etamycin

S. loTdensis (ATCC11415) vernami "; ina

S. mitakaensis (ATCC15297) mycamycin

S. ostreogriseus (ATCC27455) ostreogricin S. pristinaespiralis (ATCC25486) pristinamicin S. virginiae (ATCC13161) virginiamiein ACTINOMYCES

A.daghestanicus etamycin

More specifically, the preparation is carried out

made starting from microorganisms chosen from among

Streptomyces alborectus, Streptomyces mitakaensis,

Streptomyces pristinaespiralis, Streptomyces ostreo-

griseus and Streptomyces virginiae.

The first stage of the preparation consists

in modifying the non-selective microorganism, in

so as to increase its overall antibiotic production capacities and / or so that it synthesizes only uroscilodaide constituents of streptogramins. This can be achieved by genetic modifications (for example, mutation at the level of structure genes of enzymes involved in the biosynthes pathway or at the level of sequences that allow the expression of such structure genes), or biochemical (modification of a mechanism of post-translation, alteration of a retroinhibition mechanism, etc.). Several mutagenic agents were used:

- physical agents: X rays, ultraviolet rays; or - chemical agents: alkylating agents such as stilmethanesulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (Delie et al. Mutation Res. 9 (1970) 167-182), or 4-nitroquinolin-1-oxide (NQO); balancing agents; intercalating agents; or

- any system of insertion of mutagenesis on ADN and in particular transposons, integrative plasmids, phages or prophages; or also - the fusion of protoplasts (Cohen, Nature 268 (1977) 171-174).

These agents (alone or in combination) can be applied on non-selective microorganisms in the state of spores, germinated spores or during germination or on the mycelium.

In the preparation it is also possible to carry out manipulations (random or targeted) which allow to obtain microorganisms capable of selectively producing a component of streptogramins starting from non-selective microorganisms.

The second stage of preparation concerns the identification and isolation of selective microorganisms. This stage can be achieved in particular by carrying out a sensitivity test for a germ. There are different germs sensitive in particular to the components of group A or to those of group B of streptogramins: for example Bacillus subtilis (ATCC6633), Bacillus circulans, Bacillus cereus (Watanabe, J. Antibio. Ser.

A XIII (1) (I960) 62), or C. xerosis (Watanabe mentioned above) which are sensitive in particular to the components of group B; Streptococcus agalactiae B96 (Antimicrob. Agents Chemother. 10 (5) (1976) 795), Micrococcus luteus (Prikrylova above) or Sarcina lutea (ATCC9341) which are specifically sensitive to components of group A. It is also possible to artificially prepare specifically sensitive germs to a component of streptogramins by inserting, in a germ sensitive to the two components of streptogramins, a gene resistant to one of them. Some of these genes have been cloned (Le Goffic et al., J. Antibio. XXX (8), 665 (1977); Le Goffic et al., Ann. Microbiol. Inst.

Pasteur 128B, 471 (1977); Solh et al., Path. Biol. 32 (5), 362, (1984), and some of these genes are introduced into different germs by adopting classical techniques of molecular biology. The selection step can also be carried out by means of the ELISA test using specific antibodies of components A or B, or also by adopting analytical techniques such as chromatography (liquid chromatography, thin layer chromatography, etc.).

In the case of a susceptibility test against a germ, it is also preferable to validate the selection by chromatographic assay.

Thus, according to the invention, it is now possible to obtain in industrial quantities a new purified form of streptogramin whose impurity content, whose definition and constancy of composition comply with the requirements of the legislation concerning registration and which also possesses an activity in vivo and improved bioavailability and also lower toxicity. The new association will thus be able to remedy the absence of treatment with an antibacterial of this class in many countries.

The new association of a component of group B of streptogramins of general formula (I) and of a component of group A of streptogramins of general formula (II) shows a particularly interesting in vivo activity, in particular on Gram-positive germs. In vivo, in mice it was shown to be active on Staphylococcus aureus IP 8203 at oral doses of 30-50 mg / kg.

By way of example, the oral CD50 values of several combinations of components of general formula (I) and (II) in the experimental rat infection caused by Staphylococcus aureus IP 8203 are reported below.

In the following table I, the studied associations are prepared by co-precipitation in hexane, starting from a solution of the components of general formula (I) and (II) in methylisobutylketone or in acetone:

egociazkne: Product (I) / product (II): CD ^ (mg / kg <)> p.o.

PI (example U / PIIB (example 18)

10/90 44

20/80 32

30/70 30

70/30 30

80/20 30

90/10 50

Table I.

In Table II below, the associations illustrated are co-crystallized products prepared as described in the examples.

Take care: Product (I) / iz edotta (Π) 3⁄4 (n »3⁄4qs) p.o. ocrystallized

PL / ΡΏΒ (example 9) 33

FTA / PHB (example 11) 2B

ΡΣΒ ^ ΡΠΒ (example 12) 32

HIC / ΡΠΒ (hexipoo 13) 36

exociartme: Product (I) / product (Π) or crystallized L <(> ng'Hg) Ρ · Ο.

ΡΊΕ / ΡΙΙΒ (eaarpio 14) I know

Factor S / ΡΏΖ (example 15) 32

attere SL / ΡΠΒ (example 16) SD

Patte S / PHF (example 17) 90

TableΠ

In Table III below, the described association is prepared in the form of a physical mixture of the powders.

A3⁄4r3⁄4d action: Phxbto (D / product (II) °° 50 (, n8 / kg: i p '° PIA (example U / PIIB (example 18) 30/70 36

PIA (example D / PIIB (example 18) 50/50 40

Factor S (example 5) / PIIB (example 18) 44

30/70

Table III

Furthermore, the new association does not show toxicity: no signs of toxicity occur in mice at the dose of 150 mg / kg orally (2 administrations).

According to the invention, when the co-crystallized association is used as a purification means of the component of general formula (II), this can be obtained by acid extraction of a solution of the co-crystallized compound in a ketone (for example, methyl isobutyl ketone), then isolation by extraction of the component of group A by carrying out a precipitation starting from the organic phase.

The following examples, given without limitation, illustrate the present invention.

In the following examples it is understood that the titles are indicated in% by weight.

and purification of Group B components Example 1

30 kg crude pristinamycin [pristinamycin IA (PIA): 20.7%, pristinamycin IB (PIB): 3.9%, pristinamycin IC (PIC): 0.6%, pristinamycin ID (PID): 0.3%, pristinamycin IIB (PIIB): 8%, pristinamycin HA (PIIA): 45%, pristinamycin IIF (PIIF): less than 0.5% (undetermined), pristynamycin IIG (PIIG): less than 0.5% (undetermined) they are suspended in 210 liters of ethyl acetate and stirred for 15 hours at room temperature. The suspension is filtered and the ethyl acetate filtrate is collected and extracted with

2 times 20 liters of sulfuric acid IN then 20

liters of distilled water. The combined aqueous phases are washed with 6 times 15 liters of ethyl acetate, then they are adjusted to pH 7 by adding 30 liters of a 10% solution of sodium bicarbonate and extracted with 3 times 30 liters of methylene chloride . The chloromethylene phases are combined and washed with 10 liters of distilled water. The methylene chloride is then distilled and replaced with 50 liters of ethanol. The mixture is then refluxed with 0.8 kg of L3S black for 30 minutes. After filtration and washing with 2 times 5 liters of ethanol, the mixture is cooled to 10 <e> C in 15 hours. After maintaining it for 1 hour at 10 ° C, the suspension is filtered and washed with 3 times 7 liters of ethanol. After drying the solid at 40 ° C under reduced pressure, 5.7 kg of purified pristinamycin I (hereinafter referred to as PI) are obtained.

Titer: 96.8 * (PIA: 81.1%, PIB: 12%, PIC: 2.6%, PID: 1.1%);

Yield in PIA: 74 *.

1500 g of purified PI are taken up with 9 liters of dichloro-1,2 ethane, then they are added with 1.5 equivalents of succinic anhydride and with 0.015 equivalents of dimethylaminopyridine. The solution is kept for 1 week at 20 ° C, then it is introduced into a column containing 10 kg of silica (20-45 micrometers) [column height: 1 m; diameter: 20 cmj. Elution is carried out by percolating a mixture of dichloro-1,2 ethane / methanol at a flow rate of 18 liters / hour for 6 hours; the percentage of methanol (having a water content of 5%) is increased from 0 * to 43⁄4> in the course of chromatography. 47 fractions of 2.4 liters are isolated.

The fractions 5 to 15 are combined, the dichloro-1,2 ethane is evaporated and replaced with 5 liters of ethanol. After crystallization, 365 g of PIA with 99.8% titer are obtained.

Example 2

The fractions 36 and 39 of the chromatography described in Example 1 are combined and the dichloro-1,2 ethane is distilled; 210 g of solid are thus obtained. 40 g of this solid are taken up with 8 liters of water to which 8 era of 10 N hydrochloric acid are added. After 3 hours at 90 ° C, the solution is neutralized to pH 6.5 with sodium bicarbonate. The solution is extracted with three times 1 liter of ethyl acetate and the extract is washed with 2 times 0.2 liters of water. After treatment with carbon, the ethyl acetate is evaporated and replaced with 600 era of ethanol. After recrystallization, 20 g of PIB with 97% titer are obtained.

Example 3

The fractions from 22 to 26 of the chromatography described in Example 1 are combined and the dichloro-1,2 ethane is distilled; 139 g of solid are thus obtained. This solid is taken up with a minimum LTI of dichloro-1,2 ethane and is introduced into a silica column. The elution is carried out by percolating a dichloro-1,2 mixture

ethane / methanol with a flow rate of 18 liters / hour for 6 hours; the percentage of methanol (containing 5 * of water) is increased from 0% to 5% during the chromatography. 48 fractions of 2.4 liters are isolated. The fractions 38 to 43 are evaporated and the solid is taken up with 300 era of ethanol. After recrystallization, 22 g of PI at 40% of PIC are obtained.

Subsequent chromatographs on silica (20-45 micrometers) by percolating with an eluent methylene chloride / methanol (98/2 volumes) allow to obtain 5 g of a solid which, after shaking with methyl-isobutyl ketone and recrystallization in ethanol, title * , 95% of PIC.

Example 4

1000 g of PI obtained as previously described in Example 1, are dissolved in the minimum quantity of chloroform and purified by successive fractions on a silica column (20-45 micrometers). After elution with chloroform containing 2-5% of methanol, a product is obtained which is concentrated to dryness. This product is then purified with 2 successive passages on the Diaion resin column percolated with an acetonitrile / water mixture (60/40 by volume). The fractions are checked by chromatography. The PID-containing fractions are pooled and concentrated to dryness. In this way about 3 g of product titrated at 60% of PID are obtained. Additional purification is carried out by counter-current chromatography using the solvent mixture methylisobutylketone / acetone / formic acid (40/2/40 by volume). The dry concentration of the fractions containing PID allows to obtain X g of a solid which titrates at 95% of PID.

Example 5

400 g of Staphylomycine (in tablet form - initial composition: virginiamycin SI (SI): 3.4%, virginiamycin S4 (S4): 0.9%] are introduced into 4 liters of water.

The tablets are disaggregated by shaking for 15 minutes at 20 ° C. 1 liter of methylene chloride is added and stirring is continued for 1 hour. The chloromethylene phase is then decanted and filtered, then it is poured, in 30 minutes, into a volume of 5 liters of hexane subjected to stirring. After 1 hour of stirring, the suspension is filtered and a solid is isolated which is washed with 3 times 250 cm of hexane. After drying, 52 g of solid product are isolated which is suspended in 370 cm of ethyl acetate. Two successive shakes are carried out at 20 ° C and with a duration of 18 hours. The filtrate corresponding to each shake is brought to dryness, then it is dissolved in 850 cm of methanol under reflux. After a gradual decrease in temperature down to -20 ° C in 16 hours, a solid is collected by filtration and washed with a small quantity of methanol. After drying the solid at 35 ° C under reduced pressure, 9 g of the S factor are obtained

(virginiamycin S).

Title: 96 # (YES: 75.4 #, S4: 20.6 #).

Yield in factor SI (virginiamycin SI): 50 #.

Example 6

1 g of factor S obtained as previously described in example 5, dissolved in acetonitrile up to a concentration of 125 mg / cm, is purified in 4 operations by column chromatography of Nucléosil5C8 (height 25 cm, external diameter 2.54 cm) by injecting a volume of 2 cm and eluting with the 60/40 water / acetonitrile mixture by volume, with a flow rate of 7.5 cm ^ / minute. Each time, a volume of 120 cm is collected with the SI factor, that is, in all, 480 cm. The chromatography is repeated four times in order to treat the totality of 1 g of factor S. A volume of about 500 cm containing the factor SI is thus collected. The acetonitrile is removed by means of a rotating evaporator. The aqueous phase is extracted with three times 50 cm of dichloromethane. The phases are gathered in chloromethylene, washed with 50 cm of distilled water, anldrified on sodium sulphate and filtered. The dichloromethane is removed by means of a rotating evaporator, at reduced pressure (5 mm of mercury). This is how they are obtained

0.67 g of SI factor titling to 99.6%.

PREPARATION OF CRUDE COMPOUNDS OF GROUP A:

Example 7

500 g crude pristinamycin (pristinamycin IA (PIA): 20.7%,

pristynamycin IB (PIB): 3.9%, pristynamycin IC (IOP): 0.6%,

pristinamycin ID (PID): 0.3%, pristinamycin IIB (PIIB): 8%, pristinamycin HA (PIIA): 45% J are dissolved in 50 liters of methyl isobutyl ketone. This solution is extracted 5 times with an aqueous phase consisting of 2.5 liters of water and 2.5 liters of IN sulfuric acid, then it is washed with three times 10 liters of water. The methyl isobutyl ketone is then treated with 7.5 liters of an aqueous solution of sodium bicarbonate at 35 g / liter, then washed with 5 liters of water. Each time, the aqueous phase is mixed with the organic phase, decanted and separated.

The organic phase obtained is put in contact with 750 g of alumina, filtered, concentrated up to a volume of about 4 liters and taken up with 5 volumes of hexane. The precipitate obtained is filtered and dried. 300 g of product are obtained which are suspended in 1 liter of Isopropanol. After stirring at 55 ° C for 45 minutes, it is filtered at 4 ° C. The filtration mother liquors are concentrated to dryness, they are taken up with 500 cm of methylisobutylketone on which 5 volumes of hexane are poured. The precipitate is filtered, washed with hexane and dried at 40 ° C under reduced pressure.

69 g of crude PIIB are obtained containing 36% of PIIB, 6% of PIIA and which no longer contains PIA.

Example 8

60 g of crude PIIB obtained as previously in Example 7 are purified in several operations, by chromatography on a Nucléosil5C8 column (column diameter 5 cm, height 30 cm) by percolating with a water / acetonitrile 60/40 eluent. Thus 250 mg of pristinamycin IIF (PU F) are obtained.

PREPARATION OF A CRYSTALLIZED PRODUCT:

In the following examples, it has been pointed out that the diffraction spectrum X of the oo-crystallized product is different from the spectrum of the component of group B crystallized, alone, in the same solvent, when it exists.

Example 9

The crude PIIB obtained previously in Example 7 is dissolved in 190 cm of acetone. 33 g of purified PI are added (PIA: 81.196, PIB: 1296, PIC: 2.696, PID: 1.196). After 17 hours of stirring at 20 ° C, a suspension is obtained which is filtered at 4 ° C. The product is washed and dried. After a recrystallization at 100 g / 1 in acetone, 10 g of white crystals are obtained whose title in PIIB + PIIF + PIIG is 5596 and whose title in PIA + PIB + PIC + PID is 43%.

Example 10

250 mg of purified PIIB, obtained as described below in Example 18, are dissolved in 17 cm of ethyl acetate. 300 mg of purified PI are added (PIA: 81.1%, PIB: 12%, PIC: 2.6%, PID: 1, 1%). After 20 hours of stirring at 20 ° C, filtration, washing and drying, 125 mg of white crystals are obtained.

Title in PIIB + PIIF + PIIG: 56%; of which PIIB 54% ,, Security in PIA + PIB + PIC + PID: 43%.

Example 11

560 mg of pure PIIB obtained as described below in Example 18, are dissolved in 5 cm of acetone. 480 mg of PIA (title 99.8%) are added. The mixture is stirred for 20 hours at 20 ° C and the suspension is filtered. After washing with 1 cm of acetone and drying for 30 hours at 40 ° C under reduced pressure (<1 kPa), 590 mg of white crystals are obtained.

Title in PIIB + PIIF + PIIG: 56%; of which PIIB 54%.

Title in PIA; 43%.

The mother liquors of the previous crystallization are taken up and added with a new charge of 560 mg of PIIB. The PIIB / PIA weight ratio is then close to 4. After 20 hours of stirring, after filtration, washing and drying, 195 mg of crystals are obtained having a degree of purity and a composition identical to those of the crystals obtained from the first jet.

Example 12

Operating as described above in Example II, but replacing PIA with 480 mg of PIB (97% titre) 820 mg of white crystals are obtained, whose PIIB + PIIF + PIIG titre is 56% (of which PIIB 54%) and the stock in PIB is 43%.

Example 13

Operating as described above in Example 11, but using 680 mg of PIXB and 580 mg of P1C (ti-3

95%) in 4 cm of acetone, 315 mg of white crystals are obtained whose title in PIIB + PIIF + PIIG is 57% (of which PIIB 55%) and whose title in PI is 42% (of which 37% PIOUS).

Example 14

Operating as described above in Example 11, but replacing PIA with 480 mg of PID (95% titre), 475 mg of white crystals are obtained, whose PIIB + PIIF + PIIG titre is 55% (of which PIIB 53%) and whose PID title is 39%.

Example 15

Operating as described above in example 11, but using 450 mg of PIIB and 360 mg of factor S (SI: 75.4%, S4: 20.6%) in 4 cm of acetone, 550 mg of white crystals are obtained. whose title in PIIB + PIIF + PIIG is 58% (of which PIIB 56%) and whose title in factor S is 41% (of which 37% of SI). Example 16

By operating as described above in example 11, but replacing PIA with 480 mg of factor SI, 750 mg of white crystals are obtained, whose titre in PIIB + PIIF + PIIG is 58% (of which PIIB 54%) and whose titre in SI factor it is 41%.

Example 17

Operating as described above in Example 11 but using 224 mg of PU F and 192 mg of

2

S factor in 2 cm of acetone, 220 mg of white crystals are obtained, whose title in PU F is 55% and the title in factor S is 39% (of which the factor SI: 31%, factor S4: 5%).

X diffraction diagrams of the products of

examples 9 to 17

The relative intensities of the main lines are shown in Table IV below. The X diffraction patterns are made on the Phillips PW1700 diffractometer with cobalt anti-cathode. Reference 100 is indicated for the

line at 15.8 A. The relative values are evaluated by measuring the height of the line, having subtracted the solid background.

Table IV

The X diffraction patterns are similar, whatever the co-crystallized product (the inter-lattice distances of the main lines are not significantly different).

PURIFICATION OF A COMPONENT OF GROUP A

Example 18

9.3 g of the product obtained in example 9 are dissolved in 490 cm of methyl isobutyl ketone. This solution is extracted twice with

370 cm of an aqueous solution of 0.5 N sulfuric acid, then it is washed with twice 150 cm of water. The organic phase is then concentrated up to a volume of about 80 cm which is poured onto 5 volumes of hexane. The precipitate obtained is washed, filtered and dried. To eliminate the methylisobutylketone, it is then taken up in acetone at 100 g / l, poured over 10 volumes of hexane, washed and dried. 3.5 g of a purified PIIB-containing product and no longer containing PI are obtained.

PIIB + PIIF + PIIG title: approximately 95% of which 92% of PIIB.

The present invention also relates to pharmaceutical compositions usable in human or veterinary medicine which contain, as an active product, the new purified association of streptograir.ine which comprises at least one component of group B of streptogramins associated with component of group A of general formula (II ), in its pure state or in the presence of one or more compatible and pharmaceutically acceptable diluents or adjuvants. These compositions can be used orally or topically. They can contain the associations according to the invention in the state of physical mixture of the powders, of coprecipitate or of co-crystallized.

As compositions for oral administration, tablets, pearls, pills, lyophilisate powders or granules can be used. In these compositions, the active product according to the invention can be mixed with one or more diluents or inert adjuvants such as sucrose, lactose or starch. These compositions may also contain substances other than diluents, for example a lubricant such as magnesium stearate.

The compositions for topical administration can be, for example, creams, ointments or lotions.

In human or veterinary therapy, the compositions according to the invention are particularly useful in the treatment of infections of bacterial origin, in particular serious infections caused by Gram-positive cocci: infections caused by staphylococci (in particular infections caused by methicillin-resistant staphylococci) , infections caused by streptococci (in particular on pneumococci resistant to penicillin and macrolides); they are also particularly useful

in the treatment of infections caused by Hemophilus, Moraxella catarrhalis, Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma pneumoniae, Ureaplasma urealyticum.

The compositions according to the invention can be used in particular in the treatment

upper and lower respiratory infections (e.g. treatment of lung infections), treatment of skin infections, long-term treatment of bone or joint infections, treatment or prophylaxis of endocarditis, dental and urinary surgery, treatment sexually transmitted diseases and also in the treatment of bacterial and parasite-induced opportunistic infections

which occur in AIDS and in the prophylaxis of staphylococcal risk in immunosuppressed.

In general, the doctor will determine the dosage he deems most appropriate, depending on the age, weight, degree of infection and

other characteristic factors of the subject to be treated. In general, the doses are comprised between 0.4 and 3.5 g of active product in 2 or 3 administrations per day, orally, in the case of an adult.

The following examples, given without limitation, illustrate compositions according to the invention.

Example A

According to the usual techniques, opaque pearls dosed at 250 mg of the co-crystallized PIB / PIIB association are prepared,

Example B

According to the usual techniques, opaque pearls are prepared dosed at 250 mg of the association Factor S / PIIB co-crystallized.

Example C

According to the usual techniques, tablets dosed at 384 mg of active product are prepared, having the following composition:

- PIB / PIIB (45% / 55%). . 384 mg - Hydroxypropylmethylcellulose. . 25 mg - Magnesium stearate. 35 mg - Colloidal silica. 14 mg - Starch. q.s. to 700 mg Example D

According to the usual techniques, tablets dosed at 384 mg of active product are prepared, having

the following composition:

- S / PIIB factor (45% / 55%). 384 mg - hydroxypropylmethylcellulose. 35 <m> S-magnesium stearate. 14 mg - Colloidal silica. 14 mg - Starch. q.s. to 700 mg Ing. Barzanò & Zanardo Milano S.p.A.

Claims (14)

CLAIMS 1. A purified form of streptogramin characterized by the fact of being constituted by the association of one or more components of group B of streptogramins, of general formula: in which it is a radical with a general formula: where R 'is a hydrogen atom or UJI hydroxyl radical and Y is a hydrogen atom, a methylamine radical or a dimethylamine radical, R is an ethyl radical or, when R' represents a hydrogen atom R can also represent methyl, e Ri and R ^ represent a hydrogen atom or Ai is a radical of the formula: in which R is an isobutyl radical, e Ri is a hydroxyl radical and 3⁄42 is a methyl radical, and one or more minority components of group A of streptogramins, of general formula: in which R "is a hydrogen atom or a methyl or ethyl radical, in the state of co-crystallized, co-precipitate or physical mixture of the powders. 2. A purified form of streptogramin according to claim 1 characterized by containing less than 6% impurities. 3. A purified form of streptogamine according to one of claims 1 or 2, consisting of an association, in the proportions of 10/90 to 90/10 (by weight), respectively, of one or more components of group B of streptogramins and of one or more minority components of group A of streptogramins, as defined in claim 1, in the state of coprecipitate or physical mixture of powders. 4. A purified form of streptogramins according to one of claims 1 to 3, consisting of an association of 20/80 to 80/20 (by weight) respectively of one or more components of group B of streptogramins and of one or more minority constituents of group A of streptogramins as defined in claim 1, in the state of coprecipitate or of a physical mixture of the powders. 5. A purified form of streptogramins according to claim 1 or 2, characterized in that it is a co-crystallized compound of one or more components of group B of streptogramins and of one or more minority components of group A of streptogramins as defined in claim 1, in a molar ratio of the order of magnitude of 1/2. 6. Use of an oo-crystallized compound according to claim 5 for the preparation of a purified form of streptogramins according to one of claims 1 to 4. 7. Use of a co-crystallized compound according to claim S for the purification of a minority component of group A of streptogramins as defined in claim 1. 8. Process for preparing a purified form of streptogramins according to one of claims 1 to 5, characterized in that the component (1 components) of group A of the streptogramins are co-crystallized with the component (s) of group B of the streptogramins as defined in claim 1, then, if necessary, the crystallized compound obtained with hilocentricular (yet suitable) of group A or B is associated to obtain an association in the desired proportions. 9. A purification process of a minority component of group A of streptogramins defined in claim 1, characterized in that a co-crystallized compound of this component is prepared with one or more components of group B of streptogramins, then the component (1 components) of group B by treatment in an acid medium. 10. A purified component of group A of the streptograms as defined in claim 1, when it is obtained by means of a co-crystallized compound according to claim 5. 11. A component of group A of streptogramins of general formula: in which R "is a hydrogen atom or an ethyl radical. 12. Use of one or more minority components of group A of the streptogramins as defined in claim 1, for the preparation of co-crystallized substances with one or more components of group B of the streptogramins or their derivatives. 13. Co-crystallized compounds consisting of one or more components of group B of streptogramins or of their derivatives and one or more minority components of group A of streptogramins defined in claim 1. 14. Pharmaceutical composition characterized in that it contains a purified form of streptogramins according to one of claims 1 to 5, in the pure state, or in the presence of any compatible and pharmaceutically acceptable diluent or adjuvant.